Treatment of Gaucher disease

 Gaucher disease, the most common lysosomal storage disease, is caused by mutations in the gene that encoding the lysosomal enzyme, acid-β-glucosidase (acid-beta-glucosidase, glucocerebrosidase, GlcCerase, E.C. 3.2.1.45). The most common treatment for Gaucher disease is enzyme replacement therapy (ERT), in which defective GlcCerase is supplemented with an active enzyme. The correlation between the ~ 200 mutations in GlcCerase and disease severity is not completely understood, although homozygosity for the common mutations N370S and L444P is associated with non-neuronopathic and neuronopathic disease, respectively.

Imiglucerase (Cerezyme®)
The X-ray structure of GlcCerase (Cerezyme®) was resolved at 2.0 A resolution (1ogs). The catalytic domain consists of a (beta/alpha)(8) TIM barrel, as expected for a member of the glucosidase hydrolase A family. The distance between the catalytic residues E235 and E340 is consistent with a catalytic mechanism of retention. N370 is located on the longest alpha-helix (helix 7 ), which has several other mutations of residues that point into the TIM barrel. Helix 7 is at the interface between the TIM barrel and a separate immunoglobulin-like domain on which L444 is located, suggesting an important regulatory or structural role for this non-catalytic domain. The structure provides the possibility of engineering improved GlcCerase for enzyme-replacement therapy, and for designing structure-based drugs aimed at restoring the activity of defective GlcCerase.

GlcCerase with cyclohexitol
The crystal structure of the human GlcCerase (colored yellow ) with covalently bound irreversible inhibitor cyclohexitol (conduritol-B-epoxide; CBE; shown in cyan with its hydroxyl groups are in red ) was solved (1y7v, ). This structure reveals that binding of CBE to the active site does not induce a global conformational change in GlcCerase and confirms that Glu340 is the active-site catalytic nucleophile, because the distance between the cyclohexitol C1 atom and Glu340 Oε2 is 1.43 Å. The comparison between the active sites of GlcCerase and another representative of the glycohydrolase family - plant β-D-glucan glucohydrolase (1iev, ), reveals that CBE bound with this plant enzyme adopted the "chair" conformation, while with human GlcCerase, it is observed in a "boat" conformation, with hydrogen bonds to Asn234 Oδ1 and Nδ2, Glu340 Oε1, Trp179 Nε1, and Asp127 Oδ1 and Oδ2. Only one of two <scene name='1y7v/Loops/3'>alternative conformations of a pair of flexible loops (L1: Ser345–Glu349, and L2: Val394–Asp399) located at the entrance to the active site in native GlcCerase (1ogs) is observed in the GlcCerase-CBE structure (1y7v), a conformation in which the active site is accessible to CBE (<font color='blue'>colored blue ), while these loops in <font color='magenta'>the second (closed) conformation are colored magenta. In <scene name='1y7v/L2/5'>loop 2, a major structural change is observed in the positions of <scene name='1y7v/L2/6'>Asn396 and Phe397 , and in <scene name='1y7v/L1/6'>loop 1 a more limited difference is observed in the conformations of <scene name='1y7v/L1/7'>Lys346 and Glu349. Analysis of the dynamics of these two alternative conformations suggests that the two loops act as a lid at the entrance to the active site. The movies 1 and 2 illustrate the dynamics of the movement of these two loops.

Native human acid β-glucosidase, expressed in cultured plant cells (prGCD, pGlcCerase)
Three-dimensional structure of recombinant plant-derived glucocerebrosidase (prGCD, 2v3f) consists of <scene name='2v3f/Cv/7'>3 domains. Domain I (residues 1–27 and 384–414, colored <font color='pink'>pink ) comprises a 3-stranded anti-parallel β-sheet flanked by a perpendicular amino-terminal strand. <font color='lime'>Domain II (residues 30–75 and 431–497, colored lime) consists of two β-sheets. <font color='red'>Domain III (residues 76–381 and 416–430, colored red) is a (β/α) 8 TIM barrel. <scene name='2v3f/Cv/10'>The catalytic site with molecule BTB is shown. <scene name='2v3f/Align/2'>Structural alignment of <font color='red'>prGCD (2v3f) with both <font color='cyan'>Cerezyme® (1ogs) and Cerezyme® covalently modified by an irreversible inhibitor, conduritol-B-epoxide (1y7v, colored <font color='yellow'>yellow ), revealed highly significant structural identity. The RMSD values for Cα atoms of these structures were of 0.64 and 0.60 Å, respectively. Moreover, there was strict conservation of the <scene name='2v3f/Align/3'>active site residues.

pGlcCerase with ligands
<scene name='2v3d/Al/3'>Superimposition of the structure of <font color='red'>native human acid β-glucosidase, expressed in cultured plant cells (pGlcCerase, 2v3f) on those of <font color='darkmagenta'>N-butyl-deoxynojirimycin/pGlcCerase (2v3d), <font color='lime'>N-nonyl-deoxynojirimycin/pGlcCerase (2v3e), and <font color='cyan'>isofagomine/deglycosylated Cerezyme (IFG/DG-Cerezyme, 2nsx) reveals significant structural identity, neither of these ligands causes structural changes upon binding to the enzyme. The imino sugar of <font color='magenta'>N-butyl-deoxynojirimycin <scene name='2v3d/Al/10'>(NB-DNJ) forms 7 hydrogen bonds and also makes several hydrophobic interactions with side chains of <font color='darkmagenta'>active site residues (2v3d). The crystal structure of <font color='lime'>pGlcCerase in complex with <font color='orange'>N-nonyl-deoxynojirimycin <scene name='2v3d/Al/11'>(NN-DNJ) (2v3e) is very similar to that of <font color='magenta'>NB-DNJ /<font color='darkmagenta'>pGlcCerase. The exception is that longer chain of <font color='orange'>NN-DNJ interacts with 2 additional residues Leu241 (<font color='lime'>labeled lime ) and Leu314 of symmetrically related monomer (not shown). Comparison of the structures of NB-DNJ/pGlcCerase (2v3d) and NN-DNJ/pGlcCerase (2v3e) with that of <scene name='2v3d/Nsx/2'>IFG/DG-Cerezyme (2nsx) shows that the pyranose-like ring forms a same number of hydrogen bonds with the enzyme in all three cases (2v3d, 2v3e, and 2nsx).

Velaglucerase alfa
The <scene name='2wkl/Al/4'>structural alignment of the crystal structure of <font color='red'>velaglucerase alfa (colored red) (2wkl) reveals that it is very similar to those of the recombinant GlcCerase produced in Chinese hamster ovary cells (<font color='blueviolet'>imiglucerase, Cerezyme®, colored blueviolet, 2j25) and in transgenic carrot cells (prGCD, 2v3f). <scene name='2wkl/Al/13'>Superposition of the two individual molecules in the asymmetric unit of velaglucerase alfa and imiglucerase demonstrates striking similarity between positions of <font color='orange'>catalytic residues E235 and E340 (colored orange) in all 4 molecules. The position of H311 is also very similar in all 4 molecules, whereas the conformations of 3 other active site residues W312, Y313, and, especially N396 are somewhat different. The active site residues (except <font color='orange'>E235 and E340 ) of the two individual molecules in the asymmetric unit of velaglucerase alfa are colored: <font color='red'>subunit A (red), <font color='lime'>subunit B (lime) and of imiglucerase: <font color='blueviolet'>subunit A (blueviolet) , <font color='magenta'>subunit B (magenta). Imiglucerase and pr-GlcCerase contain a <scene name='2wkl/Al/14'>histidine at residue 495 <font color='blueviolet'>(blueviolet), whereas velaglucerase alfa contains <scene name='2wkl/Al/15'>arginine <font color='red'>(red). Mutations which cause Gaucher disease, <scene name='2wkl/Al/16'>R496 and D474 are close to R495 near the N-terminus of GlcCerase. The <scene name='2wkl/Al/11'>velaglucerase alfa (<font color='blue'>its glycans are colored blue ) and <scene name='2wkl/Al/12'>imiglucerase (<font color='magenta'>its glycans are colored magenta ) have different carbohydrate composition. This difference in glycosylation causes the increased cellular uptake of velaglucerase alfa over imiglucerase and could lead to improvement of treatment of Gaucher disease.

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Additional Resources
For additional information, see: Metabolic Disorders